P2X7 Receptor Antagonist Reduces Fibrosis and Inflammation in a Mouse Model of Alpha-Sarcoglycan Muscular Dystrophy

Limb-girdle muscular dystrophy R3, a rare genetic disorder affecting the limb proximal muscles, is caused by mutations in the α-sarcoglycan gene (Sgca) and aggravated by an immune-mediated damage, finely modulated by the extracellular (e)ATP/purinoceptors axis. Currently, no specific drugs are available. The aim of this study was to evaluate the therapeutic effectiveness of a selective P2X7 purinoreceptor antagonist, A438079. Sgca knockout mice were treated with A438079 every two days at 3 mg/Kg for 24 weeks. The P2X7 antagonist improved clinical parameters by ameliorating mice motor function and decreasing serum creatine kinase levels. Histological analysis of muscle morphology indicated a significant reduction of the percentage of central nuclei, of fiber size variability and of the extent of local fibrosis and inflammation. A cytometric characterization of the muscle inflammatory infiltrates showed that A438079 significantly decreased innate immune cells and upregulated the immunosuppressive regulatory T cell subpopulation. In α-sarcoglycan null mice, the selective P2X7 antagonist A438079 has been shown to be effective to counteract the progression of the dystrophic phenotype and to reduce the inflammatory response. P2X7 antagonism via selective inhibitors could be included in the immunosuppressant strategies aimed to dampen the basal immune-mediated damage and to favor a better engraftment of gene-cell therapies.


P2X7 Targeting by A438079 Improves Functional, Biochemical and Morphological Parameters in Sgca Mice
In order to evaluate the therapeutic efficacy of a selective P2X7-targeting compound in an experimental model of α-sarcoglycanopathy, we treated four-week-old male Sgca knockout mice (also termed Sgca-null) mice with A438079, a specific antagonist of P2X7 [26]. The drug was administered to Sgca-null (here from referred as Sgca) mice by i.p. injections at the dose of 3 mg/kg every other day for 24 weeks ( Figure 1a). Sgca mice injected with PBS (Sgca CTR) and Wild-Type (WT) mice served as controls.
The animals were weighted and followed once a week for signs of toxicity until the sacrifice. As shown in Supplementary Figure S1, the weight gain curve of Sgca mice treated with A438079 (Sgca A438079) was not significantly different from that of Sgca CTR mice. In addition, no signs of toxicity, including ruffled fur, vomiting, hyperactivity or loss of ambulation and breathing depression, were observed (data not shown). At the beginning (0 time) and after 6, 12, 18 and 24 weeks of treatment, animals were evaluated for muscle strength by the four-limb hanging test. Figure 1b shows that Sgca CTR mice progressively lost muscle strength up to 18 weeks (Sgca CTR vs. WT at 12 week p < 0.001, at 18 week p < 0.0001). At 24 weeks, they still showed significantly lower strength when compared to WT mice (p < 0.0001). The apparent slight recovery between 18 and 24 weeks was not significant. On the contrary, Sgca A438079 initially showed reduced functional performance, similarly to Sgca CTR mice, but after 6 weeks of treatment, muscle strength began to recover, almost reaching the performance of WT animals up to 24 weeks. The difference between Sgca CTR and Sgca A438079 mice at 12, 18 and 24 weeks was highly significant (p < 0.05 at 12, p < 0.0001 at 18 and 24 weeks). Accordingly, muscle strength in WT and Sgca A438079 mice was not significantly different at any time point. The efficacy of A438079 was confirmed by the analysis of serum levels of CK, a marker of muscle degeneration. As shown in Figure 1c, CK serum levels of Sgca CTR mice measured >10 times more than the WT levels (mean value of WT mice: 406 UI/l, n = 9; mean value of Sgca CTR mice: 5537 UI/l, n = 12, p < 0.05). Interestingly, A438079 treatment significantly reduced (by 76%) serum CK in Sgca mice after 12 weeks of treatment (mean value in Sgca A438079 mice: 1338 UI/l, n = 8 vs. Sgca CTR mice p < 0.05). After 24 weeks of treatment serum CK levels of Sgca CTR mice were 6.3 times higher than the WT levels and A438079 led to a serum CK decrease in the treated mice, although it did not reach statistical significance, likely due to increased variability of the measurements (mean value of WT mice: 829 UI/l, n = 12; mean value of Sgca CTR mice: 5235 UI/l, n = 10, p < 0.05). The animals were weighted and followed once a week for signs of toxicity until the sacrifice. As shown in Supplementary Figure S1, the weight gain curve of Sgca mice treated with A438079 (Sgca A438079) was not significantly different from that of Sgca CTR mice. In addition, no signs of toxicity, including ruffled fur, vomiting, hyperactivity or loss of ambulation and breathing depression, were observed (data not shown). At the beginning (0 time) and after 6, 12, 18 and 24 weeks of treatment, animals were evaluated for (a): Experimental design: Four-week-old male Sgca mice were treated with PBS vehicle (Sgca CTR, n = 12) and A438079 (Sgca A438079 n = 8) that was administered intraperitoneally at the dose of 3 mg/Kg every other day for 24 weeks. Age-matched male C57BL/6 Wild Type (WT n = 12) mice were used as negative control; (b): four-limb hanging test was performed before treatment and at the end of 6, 12, 18 and 24 weeks of treatment. Each value represents the mean ± SD of animals evaluated. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. Green asterisks indicate statistical significance between WT and Sgca CTR (***, p < 0.001, ****, p < 0.0001). Black asterisks indicate statistical significance between Sgca CTR and Sgca A438079 (*, p < 0.05; ****, p < 0.0001). No statistical significance (n.s.) was identified between WT and Sgca A438079 mice; (c): serum creatine kinase (CK) levels were evaluated at the end of the twelfth and the twenty-fourth week of treatment. Blood samples were obtained by retro orbital withdraw from WT, Sgca CTR and Sgca A438079 mice. Data are expressed as mean ± SD of animals evaluated. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. Asterisks indicate statistical significance (*, p < 0.05). To investigate whether the improved functional performance of Sgca A438079 mice correlated with decreased inflammation and muscle degeneration, we performed histological analysis by H&E staining of quadriceps from WT, Sgca CTR and Sgca A438079 mice.
Quadriceps from Sgca CTR mice presented areas of necrotic cells surrounded by reactive macrophage infiltration (Figure 2a) which were reduced upon A438079 treatment (Figure 2a). According to the histological analysis (Figure 2b), the percentage of centrally nucleated myofibers dramatically increased in quadriceps of Sgca mice in comparison to WT animals (p < 0.001) and was reduced by 12% in A438079-treated animals (p < 0.001) ( Figure 2c). As expected, the fiber size variability, calculated as coefficient variance Z of minimal Feret's diameter, was wider in Sgca CTR compared to WT mice (p < 0.001) but was significantly down-modulated by A438079 treatment (p < 0.05) (Figure 2d).
(by 76%) serum CK in Sgca mice after 12 weeks of treatment (mean value in Sgca A mice: 1338 UI/l, n = 8 vs. Sgca CTR mice p < 0.05). After 24 weeks of treatment seru levels of Sgca CTR mice were 6.3 times higher than the WT levels and A438079 l serum CK decrease in the treated mice, although it did not reach statistical signif likely due to increased variability of the measurements (mean value of WT mice: 82 n = 12; mean value of Sgca CTR mice: 5235 UI/l, n = 10, p < 0.05).
To investigate whether the improved functional performance of Sgca A43807 correlated with decreased inflammation and muscle degeneration, we performed logical analysis by H&E staining of quadriceps from WT, Sgca CTR and Sgca A mice.
Quadriceps from Sgca CTR mice presented areas of necrotic cells surrounded active macrophage infiltration (Figure 2a) which were reduced upon A438079 tre (Figure 2a). According to the histological analysis (Figure 2b), the percentage of ce nucleated myofibers dramatically increased in quadriceps of Sgca mice in compar WT animals (p < 0.001) and was reduced by 12% in A438079−treated animals (p < (Figure 2c). As expected, the fiber size variability, calculated as coefficient varian minimal Feret's diameter, was wider in Sgca CTR compared to WT mice (p < 0.0 was significantly down−modulated by A438079 treatment (p < 0.05) (Figure 2d).  : frozen quadriceps tissue sections from WT, Sgca CTR and Sgca A438079 mice were collected at the end of the twenty-fourth week of treatment and stained with wheat germ agglutinin (WGA) and DAPI. The whole quadricep section has been re-constructed; (c): percentage of central nuclei was quantified in four consecutive fields for each muscle section stained with wheat germ agglutinin (WGA) and DAPI and normalized for the fiber number of each field (WT n= 4; Sgca CTR n = 4; Sgca A438079 n = 4); (d): muscle fiber diameter variability from WT, Sgca CTR and Sgca A438079 mice was calculated in the whole area as variance coefficient Z of minimal Feret's diameter (WT n= 12; Sgca CTR n = 12; Sgca A438079 n = 7). In panels (c,d), data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. Asterisks indicate statistical significance (*, p < 0.05; ***, p < 0.001).

A438079 Reduces Muscular Fibrosis and Inflammation in Sgca Mice
Fibrosis as characterized by replacement of muscle tissue with collagen deposits is the histopathological hallmark of end-stage muscular dystrophies, including alfa-sarcoglycanopathy [27]. In order to establish whether A438079 might impact collagen deposits, we performed a Masson trichrome staining on quadriceps of WT, Sgca CTR and Sgca A438079 mice and evaluated the fraction area of fibrotic reactions. As shown in Figure 3a, Sgca CTR quadriceps accumulated abundant extracellular matrix deposits which were increased in comparison to WT mice (mean value of WT mice: 1.47, n = 7; mean value of Sgca CTR mice: 2.92, n = 11, p < 0.05). A438079 treatment led to a 37% reduction of extracellular matrix deposition fraction area as compared to Sgca CTR mice (mean of Sgca A438079 mice: 1.85, n = 7, p < 0.05). No significant difference was observed between WT and Sgca A438079 animals.

A438079 Reduces Muscular Fibrosis and Inflammation in Sgca Mice
Fibrosis as characterized by replacement of muscle tissue with collagen deposits the histopathological hallmark of end−stage muscular dystrophies, including alfa−sarc glycanopathy [27]. In order to establish whether A438079 might impact collagen deposi we performed a Masson trichrome staining on quadriceps of WT, Sgca CTR and Sg A438079 mice and evaluated the fraction area of fibrotic reactions. As shown in Figure 3 Sgca CTR quadriceps accumulated abundant extracellular matrix deposits which were creased in comparison to WT mice (mean value of WT mice: 1.47, n = 7; mean value of Sg CTR mice: 2.92, n = 11, p < 0.05). A438079 treatment led to a 37% reduction of extracellu matrix deposition fraction area as compared to Sgca CTR mice (mean of Sgca A4380 mice: 1.85, n = 7, p < 0.05). No significant difference was observed between WT and Sg A438079 animals.  , Sgca CTR (n = 11) and Sgca A438079 (n = 7) were collected after 24 weeks and stained with a standard Masson trichrome stain protocol. A representative image is shown. Final magnification, 20×. In the right part of the panel (a) a graph of the fraction areas of fibrotic green positive signal/fraction area (%) of total section area of muscles evaluated is shown. Data are expressed as mean ± SD. Statistical analysis was performed by one-tailed ANOVA followed by Tukey's multiple comparison test. Asterisks indicate statistical significance (*, p < 0.05); (b): frozen sections of quadriceps from WT (n = 8), Sgca CTR (n = 12) and Sgca A438079 (n = 8) were collected after 24 weeks and stained with an acid phosphatase technique. A representative image is shown. Black arrow indicates inflammatory infiltrates. Final magnification, 20×. In the right part of panel (b), a graph of the fraction areas of inflammatory red positive signal/fraction area (%) of total section area of muscles evaluated is shown. Data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. Asterisks indicate statistical significance (**, p < 0.01; ***, p < 0.001).
To evaluate the anti-inflammatory effect of A438079, quadricep sections of WT, Sgca CTR and Sgca A438079 mice were stained with acid phosphatase, which provides a red positive signal in activated macrophages and degenerative myofibers. As shown in Figure 3b, the acid phosphatase-positive area fraction of quadriceps from Sgca CTR mice was increased in comparison to that of WT mice (mean value of WT mice: 0.26, n = 8; mean value of Sgca CTR mice: 1.47, n = 12, p < 0.001). Interestingly, A438079 led to 52% reduction of In contrast, no significant difference was observed between WT and Sgca A438079 animals.

A438079 Reduces Innate Inflammatory Cells and Increases T Regulatory Lymphocytes in Limb Muscles of Sgca Mice
In order to better characterize the phenotype of inflammatory muscle infiltrates, we performed a cytometric analysis of a pool of limb muscles, including gastrocnemius, quadriceps and anterior tibialis, isolated from WT, Sgca CTR and Sgca A438079 mice. As shown in Figure 4a, limb muscles of Sgca CTR mice were characterized by the presence of CD45 + hematopoietic immune cells which, in contrast, were not detected in WT mice (WT vs. Sgca CTR, p < 0.01).
increased in comparison to that of WT mice (mean value of WT mice: 0.26, n = 8; m value of Sgca CTR mice: 1.47, n = 12, p < 0.001). Interestingly, A438079 led to 52% reduc of inflammatory area fraction of Sgca mice (mean of Sgca A438079 mice: 0.70, n = 8 0.01). In contrast, no significant difference was observed between WT and Sgca A438 animals.
Furthermore, the analysis of the peripheral blood (PB) immune cell populations of WT, Sgca CTR and Sgca A438079 mice showed that the dystrophic animals presented significantly higher percentages of DC compared to WT animals (Figure 5c, WT vs. Sgca CTR, p < 0.001 for DC). Treatment with A438079 significantly reduced the percentage of DC, CD8 + lymphocytes and T reg cells (Figure 5c,f,g, Sgca CTR vs. Sgca A438079, p < 0.05 for DC; p < 0.001 for CD8 + lymphocytes and p < 0.001 for Treg).
Furthermore, the analysis of the peripheral blood (PB) immune cell popula WT, Sgca CTR and Sgca A438079 mice showed that the dystrophic animals presen nificantly higher percentages of DC compared to WT animals (Figure 5c, WT vs. Sg p < 0.001 for DC). Treatment with A438079 significantly reduced the percentage CD8 + lymphocytes and T reg cells (Figure 5c, f, g, Sgca CTR vs. Sgca A438079, p < DC; p < 0.001 for CD8 + lymphocytes and p < 0.001 for Treg).  No significant changes were observed in the spleen of WT, Sgca CTR and Sgca A438079 mice, with the only exception of CD3 + T lymphocytes, which were slightly increased in Sgca A43879 mice vs. Sgca CTR (Supplementary Figure S2e, Sgca CTR vs. Sgca A438079, p < 0.01).

Discussion
In the present study, we provide evidence that the pharmacological inhibition of P2X7 by the selective antagonist A438079 attenuated the dystrophic phenotype of Sgca mice by reducing fibrosis and inflammation and improved muscle performance. P2X7 is an ATP receptor belonging to the ionotropic purinergic P2X subfamily, which is expressed on virtually all cell types of the immune system and regulates the innate and adaptive immune responses [28]. Alongside its expression on immune cells, P2X7 expression and function are upregulated in the dystrophic muscle [18,21,25,[29][30][31]. In this context, we and other groups demonstrated that the genetic ablation of P2RX7 and its pharmacological inhibition by oATP, an irreversible, broad-spectrum P2X7 antagonist [17], produced significant improvements in key functional and molecular disease parameters in mdx and Sgca mice [18,24,25,30]. However, oATP can also interact with other P2X receptors, including P2X4 [32][33][34], and appears to exert anti-inflammatory effects, modulating the immune response independently of P2X7 blockage [33,35,36]. Therefore, experiments using oATP cannot unambiguously establish a role in inflammatory diseases for a specific member of the P2X family. Neurotoxicity has been described for oATP, likely due to the low specificity of the drug [37]. In order to overcome the above-described limitations, and to define the therapeutic effect of P2X7 targeting approaches in α-sarcoglycan-deficient muscular dystrophy, we specifically inhibited P2X7 using the A438079 molecule, one of the most potent and selective antagonists that competitively blocks P2X7 receptor in vitro activation and produces anti-nociceptive effects in in vivo settings [38][39][40]. In addition to nociception, A438079 has already been successfully used in in vivo models of hyperalgesia [41], epilepsy [42,43], Parkinson's disease [44], salivary gland exocrinopathy [45], and Charcot-Marie-Tooth 1A disease [46]. Our results clearly show that, by targeting P2X7, A438079 ameliorated functional and morphological parameters in Sgca mice. In particular, A438079 improved muscle morphology by reducing the percentage of centralized nuclei and the coefficient variance Z of minimal Feret's diameter (Figure 2c,d), which are typical signs of dystrophic damage [47]. Furthermore, according to previous studies [24], a relevant therapeutic effect exerted by A438079 was the reduction of muscle fibrosis (Figure 3a). P2X7 has been described to play a nodal role in triggering fibrosis through activation of multiple intracellular pathways that converge in inducing the collagen biosynthetic machinery in various organs [48]. As such, P2X7 blockade may interfere with the main pro-fibrotic pathways, thus possibly representing a target for the pharmacological modulation of fibrotic processes. The most evident beneficial effect of A438079 in Sgca animals was a significant modulation of muscle inflammation, a key feature of muscular dystrophies participating in the disease progression but also mediating muscle repair. The dichotomous role of inflammation has been extensively studied in Duchenne muscular dystrophy (DMD), in which CD4 + and CD8 + T cells, macrophages, eosinophils and natural killer T cells infiltrated both human and mouse dystrophic muscle [9,49]. In particular, proinflammatory monocytes CD11b + /Ly6G − /Ly6c + have been reported to be the first innate immune cells to be mobilized from the bone marrow into the circulation and recruited to the site of tissue injury [50], such as dystrophic muscles, where they differentiated into inflammatory macrophages [51,52]. Different studies demonstrated that neutrophils actively participated in the exacerbation of muscular dystrophy and their specific depletion reduced muscular necrosis and inflammation in mdx mice [53][54][55]. Moreover, neutrophil-derived elastase impaired myoblast proliferation, survival and differentiation [56]. In line with this notion, we found that A438079 caused a significant reduction of innate immune cells, including neutrophils, activated monocytes and dendritic cells infiltrating the limb muscles of Sgca mice. The downregulation of innate immune response by P2X7 blockade was also observed in dystrophic mice (mdx and Sgca) treated with other P2X7 antagonists, i.e., oATP or zidovudine (AZT) [18,25,57]. However, the mechanism underlying the latter effect is still unclear. P2X7 antagonists could directly inhibit P2RX7 expressed by inflammatory innate immune cells infiltrating the dystrophic muscle. Alternatively, these agents might reduce inflammatory cell migration into the injured tissue. In favor of the first hypothesis, Pharmaceuticals 2022, 15, 89 9 of 15 innate immune cells are known to express functional P2X7, which in turn triggers inflammasome activation [17,28,[58][59][60]. The second hypothesis is sustained by data showing P2X7-dependent release of chemotactic factors by macrophages. These factors, including CXCL2/macrophage inflammatory protein-2 (MIP-2), were involved in the recruitment of neutrophils into the injured tissue [61]. The immune phenotype of limb muscle from Sgca mice treated with A438079 also showed that the pharmacological treatment significantly increased Foxp3 + Treg without affecting CD4 + and CD8 + T lymphocytes. These findings are consistent with previous studies in which P2X7 blockade by oATP or P2X7 genetic ablation in dystrophic mice resulted in a significant increase of Foxp3 + Treg [18,24,25]. Treg have been described to play a dual beneficial role in dystrophic muscles. On one side, they suppress type 1 inflammation by secreting IL-10; on the other side, Treg may also have direct effects on muscle growth and regeneration through secretion of amphiregulin, an epidermal growth factor family member whose receptors are expressed on muscle satellite cells that are critical for muscle regeneration [62,63]. Interestingly, our results showed that A438079 exerted not only a local but also a systemic anti-inflammatory effect by reducing circulating CD8 + cytotoxic T lymphocytes in Sgca mice. Currently, physical therapy and prevention of secondary cardiac, pulmonary or orthopedic complications are the only possible care interventions. Although a chronic inflammatory response is documented in muscle specimens from LGMD R3 patients, no trials assessing the effects of immunosuppressive therapies have been proposed in alpha-sarcoglycan deficiency. However, two unrelated LGMD R3 patients treated with steroids showed clinical improvement [64][65][66]. In light of these considerations, strategies aimed at reducing muscle inflammation, increasing the amount of Treg infiltrating an injured muscle and exerting a systemic anti-inflammatory effect, i.e., antagonists of P2X7, might represent a therapeutic approach for LGMD R3. Immunomodulatory regimens become even more relevant as new gene therapies or gene editing approaches are being developed for alpha-sarcoglycanopathy [67][68][69], with gene therapy being in Phase I/II clinical trials for LGMD R3 patients (NCT01976091; NCT00494195). Since the success of gene therapy in the muscle tissue has to challenge the pre-existing status of chronic tissue inflammation typically identified in this diseases [67][68][69][70][71], our data suggest that P2X7 antagonism might represent a good strategy to dampen chronic inflammation, possibly leading to a better delivery of gene therapy. To date P2X7 selective antagonists have already been tested in Phase I/II clinical trials for the treatment of Crohn's disease, rheumatoid arthritis, basal cell carcinoma with an overall good tolerability and variable efficacy [72] and a new trial is currently ongoing assessing the effects of JNJ-54175446, a potent, brain-penetrant, selective P2X7 antagonist [73] in patients with major depressive disorder (ClinicalTrials.gov Identifier: NCT04116606).

In Vivo Experiments
C57BL/6 wild-type (WT) and Sgca knockout mice (also termed Sgca-null) were bred in the Animal Facility at Policlinico San Martino, Genova. All mice were housed under standard specific pathogen-free conditions and allowed access to food and water ad libitum. All experimental protocols were approved by the Policlinico San Martino Animal Welfare Body and by the Italian Ministry of Health (Authorization n • 215-2018-PR). Sgca mice were previously described [74]. The study design defined a minimum goal of a n = 8 animals per genotype and experimental group. According amounts of the drug A438079 was acquired from Tocris Bioscience Bristol, UK. Four week old Sgca male mice were randomly divided into two groups: one treated by i.p. injections with A438079 at 3 mg/Kg every other day in the morning between 10-12 for 24 weeks (Sgca A438079) and one treated with the same volume of phosphatase-buffered saline (PBS, Sigma Aldrich, St. Louis, MO, USA) every other day for 24 weeks (Sgca Control: Sgca CTR). In consideration of the high variability of the histological markers in mouse models of muscular dystrophies [18,25], and in order to increase the statistical power of the study a n of 12 WT mice and a n of 12 Sgca CTR were included. The dose of 3 mg/kg of A438079 proved to be well tolerated by wild-type rats [46]. A438079 was reconstituted at a final concentration of 1 mg/mL in PBS and stored at −20 • C; the reconstituted drug was thawed and immediately used at a final concentration of 3 mg/Kg. All animals were euthanized at the end of treatment by carbon dioxide inhalation and muscles were collected for histological and cytofluorimetric analysis. A group of age-matched WT C57Bl/6 male mice was used as internal control. All animals were weighed and followed for any sign of toxicity, including ruffled fur, vomiting, hyperactivity or loss of deambulation and breathing depression once a week. Blood samples from Sgca CTR and Sgca A438079 mice were obtained from the saphenous vein (90 µL) before treatment and 12 and 24 weeks after treatment. The samples were centrifuged at 3600× g for 30 min and immediately after centrifugation the serum was isolated and stored at −80 • C. Serum creatine kinase (CK) levels were measured using a clinical-standard automatic chemistry analyzer (BS-380 Mindray, Milan, Italy).

Four-Limb Hanging Test
Before treatment and at the end of the sixth, twelfth, eighteenth and twenty-fourth week of treatment, the muscle strength of WT, Sgca CTR and Sgca A438079-mice was scored through the four-limb hanging test. Mice were subjected to a 180-s lasting hanging test, during which a falling score was recorded. The animals had to hang for three trials, and the average maximum hanging time of the three trials was measured (standard operating procedure, https://treat-nmd.org/research-overview/preclinical-research/experimentalprotocols-for-dmd-animal-models, last accessed on September 2021.

Histological Studies, Imaging and Analysis
Quadriceps isolated from WT, Sgca CTR and Sgca A438079 mice were cut on cryostat, and 7-µm-thick sections were stained with standard hematoxylin and eosin (H&E) (reagents from Sigma Aldrich), acid phosphatase (reagents from Sigma Aldrich) to detect inflammatory reactions and Masson trichrome (reagents from Sigma Aldrich) to evaluate muscle fibrosis. Representative pictures were taken at 20× magnification. To quantify the extension of the inflammatory response and fibrotic area, images of stained sections were acquired using a Nikon Ti Eclipse microscope equipped with a 20× objective. Whole sections were imaged with an automated tile scan acquisition (usually over a 45 mm 2 -surface) by using the perfect focus system (PFS) to control the focal plane. Quantification of acid phosphatase and trichrome staining was performed using semi-automated measurement tools in NIS-Elements AR software version 4.20 and expressed in terms of fraction area (the ratio between total section area and the area of the stained objects that were detected by HSI thresholding mode). All the histological analyses were performed blind to experimental group identity. The Masson Trichrome and acid phosphatase staining were completely negative in WT mice; therefore, the n was reduced to 7 animals.The histological sections of Sgca CTR and Sgca A438079 treated mice displaying freezing artifacts were not analyzed and are excluded from the results.

Flow Cytometry
Hematopoietic cells were collected from different districts, namely PB, spleen and limb muscles. Cells collected from PB were first incubated with 2 µL/sample of TruStainFcX TM anti-mouse CD16/32 for 5 min in order to block Fc receptor and then with cocktails of antibodies specific for CD45, CD3, CD4, CD8, CD11b, CD11c, CD25, F4/80, Ly-6C, Ly-6G and Foxp3 for 30 min. The intracellular staining of transcription factor Foxp3 was performed using the Foxp3/Transcription factor staining buffer set (Thermo Fisher) as described by the manufacturer. After antibody incubation, samples were lysed (Becton Dickinson Pharm Lyse TM, San Josè, CA, USA), washed and resuspended in 300 ul of PBS. All the antibodies were purchased from Biolegend (San Diego, CA, USA). Gastrocnemius, quadriceps and anterior tibialis excised from WT, Sgca CTR and Sgca A438079 mice were resuspended in RPMI 1640 base medium (Euro Clone, Milan, Italy), mechanically and enzimatically digested using Skeletal Muscle Dissociation Kit (Miltenyi Biotec, Bologna, Italy) and filtered through 100-and 70-µm mesh filters (BD Bioscience, San Jose, CA, USA) (Supplementary Method). After filtration, cells were purified using gradient centrifugation by Percoll solution (GE Healthcare Bio-sciences, Uppsala, Sweden) and stained with Live/Dead TM Fixable Yellow Dead Cell Stain Kit (Invitrogen, Thermo Fisher Scientific) and the antibodies listed above. The spleen from WT, Sgca CTR and Sgca A438079 mice was mechanically digested, filtered through 100-and 70-µm mesh filters, counted and stained as described for PB and muscle. All acquisitions were performed with a three laser LSR Fortessa X20 (Becton Dickinson) and obtained FSC files were analysed with Kaluza Software (version 2.1, Beckman Coulter). The immune profile of the peripheral blood, spleen and muscles was performed in the same animals (WT n = 6, Sgca CTR n = 7 and Sgca A438079 n = 5).

Statistical Analysis
Statistical parameters, including the exact value of n and statistical significance, are reported in the figures and their associated legends. Results were analyzed using a one-way ANOVA followed by Tukey's multiple comparison test or unpaired t-test, where indicated, using GraphPad Prism 3.0 software (GraphPad Software, El Camino Real, San Diego, CA, USA). Asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001).

Conclusions
In conclusion, A438079 ameliorated the dystrophic phenotype of Sgca mice by reducing muscle fibrosis and inflammation and by improving functional muscle performance. In the current scenario of clinical trials including gene therapy, selective P2X7 antagonists could represent candidates for a combinatory therapy to endorse the efficacy of disease-specific gene therapy by dampening the basal muscular inflammation.